Papillomavirus pseudoviruses for detection and therapy of tumors

ABSTRACT

Disclosed herein are methods of detecting tumors, monitoring cancer therapy, and selectively inhibiting the proliferation and/or killing of cancer cells utilizing a papilloma pseudovirus or a papilloma virus-like particle (VLP).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.13/763,365, filed Feb. 8, 2013; which is a Divisional of U.S. patentapplication Ser. No. 12/598,684, filed Feb. 8, 2010, now U.S. Pat. No.8,394,411; which is a national stage application under 35 U.S.C. 371 ofPCT Application No. PCT/US08/62296 having an international filing dateof May 1, 2008, which designated the United States; which PCTapplication claimed the benefit of U.S. Provisional Application No.61/065,897, filed Feb. 14, 2008, and U.S. Provisional Application No.60/928,495, filed May 8, 2007; the entire disclosure of each of which isincorporated herein by reference.

REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledNIH360 001VPC.txt, created Apr. 25, 2008, which is 1 Kb in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to the fields of molecular biology and medicine.More specifically, disclosed herein are methods for detecting tumors andtreating subjects suffering from cancer using papilloma pseudovirusesand virus-like particles (VLPs).

BACKGROUND OF THE INVENTION

Cancer is diagnosed in more than 1 million people every year in theUnited States alone. In spite of numerous advances in medical research,cancer remains the second leading cause of death in the United States,accounting for roughly 1 in every four deaths. Although numeroustreatments are available for various cancers, many forms of cancerremain uncurable, untreatable, and/or become resistant to standardtherapies. For example, tumors may be inoperable because of theirlocation or they may metastasize, making it difficult or impossible totreat the disease. Current therapies have considerable shortcomings. Forinstance, radiation therapy can cause damage to epithelial surfaces,swelling, infertility, fatigue, fibrosis, hair loss, dryness, andcancer. Chemotherapy can induce nausea, vomiting, diarrhea,constipation, anemia, malnutrition, hair loss, memory loss, depressionof the immune system and hence infections and sepsis, hemorrhage,secondary neoplasms, cardiotoxicity, hepatotoxicity, nephrotoxicity, andotoxicity. Clearly the need for robust techniques to diagnose and treatcancer is manifest

Viruses have been shown to have tremendous utility in a variety ofbiomedical applications. Many of these techniques take advantage of theunique ability of viruses to enter cells at high efficiency. Some ofthese applications exploit viral gene expression and replication toinduce expression of an inserted heterologous gene. It is well knownthat a variety of viruses deliver and express genes in cells (eitherviral or other genes), which may be useful, for example, in genetherapy, the development of vaccines, or cancer biology.

There is extensive literature on the use of viral vectors, particularlythose based on adenovirus, adeno-associated virus (AAV), herpes virusand retrovirus, to increase the potency of anti-tumor therapy, however,these methodologies are in their infancy.

SUMMARY OF THE INVENTION

Embodiments disclosed herein relate to methods for detecting thepresence of cancer cells, (e.g., a tumor cell), bound to at least onepapilloma pseudovirus (PsV) or papilloma virus-like particle (VLP). Someapproaches involve identifying a subject having or suspected of havingcancer cells, administering to the subject a detectable amount of apapilloma pseudovirus or VLP that comprises a detectable label, anddetecting the presence or absence of cancer cells bound to the papillomapseudovirus or VLP that comprises the detectable label. In someembodiments, the label is chemically coupled to the pseudovirus or VLP.In other embodiments, the presence, absence, or amount of papillomapseudovirus or VLP bound to cancer cells and the presence, absence, oramount of papilloma pseudovirus or VLP bound to normal cells ismeasured. In more embodiments, the pseudovirus comprises a gene encodinga label (e.g., luciferase or GFP). Other labels, including fluorescent,radioactive, or chemiluminscent labels, which can be incorporated in orcoupled to the PsV or VLP, are also contemplated for use with someembodiments.

Further embodiments disclosed herein relate to methods for monitoring acancer therapy in a subject including identifying a subject with acancer, providing the subject a cancer therapy, administering to thesubject a detectable amount of a papilloma pseudovirus or VLP thatcomprises a detectable label, and determining the presence or amount ofPsV or VLP bound to cancer cells in the subject after, or during thecourse of the treatment with the cancer therapy. By using successiveinoculations with fluorescently labeled PsV or VLP, for example, realtime efficacy of the particular therapy over time can be evaluated. Insome embodiments, the label is chemically coupled to the pseudovirus orVLP. In other embodiments, the presence or amount of papillomapseudovirus or VLP bound to the cancer cells and the presence or amountof papilloma pseudovirus or VLP bound to normal cells is measured. Insome embodiments, the pseudovirus includes a gene encoding the label or,optionally, a therapeutic nucleic acid (e.g., an oligo T nucleic acid).

More embodiments disclosed herein relate to methods of selectivelyinhibiting the proliferation of cancer cells and/or killing cancer cellswithout inhibiting proliferation of and/or killing normal cellsincluding identifying a subject with a cancer and administering to theidentified subject an inhibitory amount of a composition comprising apapilloma pseudovirus or VLP and a therapeutic agent. In someembodiments, the therapeutic agent is chemically coupled to thepapilloma pseudovirus or VLP. In other embodiments, the therapeuticagent is incorporated within the papilloma pseudovirus or VLP. In someembodiments, the therapeutic agent is a toxin. In some embodiments, thetherapeutic agent comprises an oligo T nucleic acid. In someembodiments, the therapeutic agent comprises a radionuclide. Additionalembodiments disclosed herein relate to kits that include a papillomapseudovirus or VLP, pharmaceutical carriers, and instructions for usingthe kit components.

Accordingly, aspects of the invention concern methods of detecting thepresence of cancer cells bound to a papilloma pseudovirus or a papillomaVLP comprising identifying a subject having or suspected of havingcancer cells; administering or providing to said subject a detectableamount of a papilloma pseudovirus or a papilloma VLP that comprises adetectable label; and detecting the presence of cancer cells bound tosaid papilloma pseudovirus or said papilloma VLP that comprises adetectable label. In some embodiments, the label is chemically coupledto said pseudovirus or VLP or said pseudovirus comprises a gene encodingsaid label and in more embodiments the presence or amount of pseudovirusor VLP bound to said cancer cells and the presence or amount ofpseudovirus or VLP bound to normal cells is measured. The label used inthese embodiments can be fluorescent, radioactive or chemiluminescent orotherwise detectable.

Aspects of the invention also include methods for evaluating a cancertherapy comprising identifying a subject with a cancer; providing saidsubject a cancer therapy; administering or providing to said subject adetectable amount of a papilloma pseudovirus or papilloma VLP thatcomprises a detectable label; and determining the presence or amount ofsaid pseudovirus or said VLP bound to cancer cells in said subject,before a treatment with said cancer therapy and during or after a periodof said treatment. In some embodiments, the label is chemically coupledto said pseudovirus or said VLP or said pseudovirus comprises a geneencoding said label and in some embodiments, the presence or amount ofsaid pseudovirus or said VLP bound to said cancer cells and the presenceor amount of said pseudovirus or said VLP bound to normal cells ismeasured. The label used can be fluorescent, radioactive,chemiluminescent or otherwise detectably labeled.

Aspects of the invention also include methods of inhibiting theproliferation of cancer cells and/or killing cancer cells withoutinhibiting proliferation and/or killing of normal cells comprisingidentifying a subject with a cancer; and administering or providing tosaid identified subject a composition that comprises a therapeutic agentformulated with a papilloma pseudovirus or a papilloma VLP. In someembodiments, the therapeutic agent is chemically coupled to saidpseudovirus or said VLP and in other embodiments the therapeutic agentis incorporated within said pseudovirus or said VLP. The therapeuticagent can be a toxin, radionuclide, ganciclovir or acyclovir, or oligoT, preferably, oligo T, of less than or equal to 200, 175, 150, 125,100, 95, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10nucleotides. In some embodiments, the therapeutic agent is a nucleicacid expressing oligo T and said nucleic acid is operably joined to aPol III promoter. In some embodiments the methods above are usedinhibit, kill, evaluate, or diagnose the status of a cancer is selectedfrom the group consisting of leukemia, lymphoma, myeloma, plasmacytoma,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat glandcarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, neuroglioma, and retinoblastoma.

Still more embodiments include kits comprising a papilloma pseudovirusor a papilloma VLP, a pharmaceutical carrier, and instructions for usingthe kit components and method of detecting the presence of cervicalcancer in a subject, comprising providing to said subject a compositioncomprising a papilloma VLP coupled to or containing a label; removingunbound VLPs that comprise said label; and detecting the presence ofcancer cells bound to said VLP that comprises said label.

In some of these embodiments, the label is chemically coupled to saidVLP and in some embodiments, the presence or amount of said VLP bound tosaid cancer cells and the presence or amount of said VLP bound to normalcells is measured. The label can be fluorescent, radioactive,chemiluminescent or otherwise detectably labeled.

Aspects of the invention also include a composition comprising a nucleicacid that comprises an oligo T domain of at least 10 and less than orequal to 200 consecutive T residues, such as an oligo T domainconsisting essentially of 45 nucleotides or an oligo T domain thatconsists of 45 nucleotides. These nucleic acids or nucleic acidsencoding these molecules can be operably linked Pol III promoter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1d . Effects of mechanical disruption, N-9 and carrageenan onHPV16 pseudovirus infection of the mouse cervicovaginal mucosa.Multispectral imaging results (representative of two or three separateexperiments), expressed as mean signal per pixel, for mice (six pergroup) are indicated on the Y axis and gels used to prepare thepseudovirus inoculum are indicated on the X axis. Method of pretreatmentis indicated by the key. Error bars represent standard error of themean. (FIG. 1a ) Comparison of the potentiation of infection bymechanical and chemical disruption. (FIG. 1b ) Protection provided bycarrageenan when mixed with the inoculum. (FIG. 1c ) Protection providedby over-the-counter lubricants when mixed with the inoculum. (FIG. 1d )Protection provided by carrageenan when mixed with N-9 duringpretreatment.

FIG. 2a-2c . Quantitative analysis of murine reproductive tractinfection. Conceptrol-treated mice were mock infected (top) orchallenged with HPV-16-tdTomato pseudovirus (bottom). After 3 d, theentire reproductive tract was dissected out and the ventral wall of thevagina and cervix incised sagitally. (FIG. 2a ) Composite Maestro image(mucosal epithelium facing up) with unmixing algorithm applied. Redsignal represents location of infection compared to backgroundautofluorescence. (FIG. 2b ) Unmixed tdTomato signal converted tograyscale. Outline of tissue denotes ROI. (FIG. 2c ) ImageJ analysis.Mean signal per pixel within the ROI was computed.

FIG. 3. The mouse intact genital tract was completely resistant toinfection after deposition of 10⁷ pseudoviral infectious units into thevagina or endocervical canal.

FIG. 4. Green fluorescent dye-coupled HPV capsids bound neither thesquamous or simple epithelium that lines the female mouse reproductivetract.

FIG. 5. Epithelial tumor cell lines were permissive for HPV5 and 16pseudovirus infection.

FIG. 6. Non-epithelial tumor cell lines were permissive for HPV5 and 16pseudovirus infection.

FIG. 7. Experimental design to test whether papilloma pseudovirusespreferentially infect tumor cells in a SHIN3-dsr peritoneal tumormetastasis model.

FIG. 8. HPV16 pseudovirus efficiently and selectively infects ovariancancer cells implanted on the peritoneal membrane as demonstrated bymultispectral fluorescence imaging.

FIG. 9. Experimental design to test whether papilloma pseudovirusespreferentially infect tumor cells in a SKOV3 peritoneal tumor metastasismodel.

FIG. 10. HPV16 pseudovirus efficiently and selectively infects ovariancancer cells implanted on the peritoneal membrane as demonstrated bymeasuring luciferase activity.

FIG. 11. HPV 16 pseudovirus efficiently and selectively infects ovariancancer cells implanted on the peritoneal membrane as demonstrated bymultispectral fluorescence imaging.

FIG. 12. HPV16 pseudovirus efficiently and selectively infects ovariancancer cells implanted on the peritoneal membrane as demonstrated bymultispectral fluorescence imaging.

FIG. 13. HPV16 pseudovirus efficiently and selectively infects lungmetastases as demonstrated by multispectral fluorescence imaging.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is the unexpected discovery that papillomapseudoviruses and papilloma VLPs selectively bind to and infect cancercells but not normal cells. While not wishing to be bound to anyparticular theory or creating an estoppel thereby, it is contemplatedthat, in comparison to current viral gene transfer vectors, papillomapseudoviruses and VLPs unexpectedly offer many benefits. Papillomapseudoviruses and VLPs will not be become involved in competinginteraction with normal cells, which can hinder the effective deliveryof the viral vectors to the cancer cells. The inability of papillomapseudoviruses and VLPs to attach to normal cells in intact tissues(e.g., untransformed or non-cancerous) will also minimize cytotoxicityof the treatment. Further, because the pseudoviruses or VLPspreferentially kill cancer cells, they will preferentially induce animmune response against the cancer cells. Lastly, pseudoviruses and/orVLPs for many papillomavirus types can be rapidly generated andpapillomavirus neutralizing antibodies are type-restricted. Accordingly,neutralizing antibody-mediated inhibition and boosting with homologouspapilloma pseudovirus or VLP can be overcome by use of papillomapseudovirus or VLP of another type.

As described herein, it is intended that where a range of values isprovided, it is understood that each intervening value, to the tenth ofthe unit of the lower limit unless the context clearly dictatesotherwise, between the upper and lower limit of that range and any otherstated or intervening value in that stated range is encompassed withinthe embodiments. The upper and lower limits of these smaller ranges mayindependently be included in the smaller ranges is also encompassedwithin the embodiments, subject to any specifically excluded limit inthe stated range. Where the stated range includes one or both of thelimits, ranges excluding either both of those included limits are alsoincluded in the embodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the embodiments belong. Although any methods andmaterials similar or equivalent to those described herein may also beused in the practice or testing of the embodiments, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “amethod” includes a plurality of such methods and reference to “a dose”includes reference to one or more doses and equivalents thereof known tothose skilled in the art, and so forth.

In some contexts, the terms “individual,” “host,” “subject,” and“patient” are used interchangeably to refer to an animal that is theobject of treatment, observation and/or experiment. “Animal” includesvertebrates and invertebrates, such as fish, shellfish, reptiles, birds,and, in particular, mammals. “Mammal” includes, without limitation,mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows,horses, primates, such as monkeys, chimpanzees, and apes, and, inparticular, humans.

In some contexts, the terms “ameliorating,” “treating,” “treatment,”“therapeutic,” or “therapy” do not necessarily mean total cure orabolition of the disease or condition. Any alleviation of any undesiredsigns or symptoms of a disease or condition, to any extent, can beconsidered amelioration, and in some respects a treatment and/ortherapy. Furthermore, treatment may include acts that may worsen thepatient's overall feeling of well-being or appearance.

The term “therapeutically effective amount/dose” or “inhibitory amount”is used to indicate an amount of an active compound, or pharmaceuticalagent, that elicits a biological or medicinal response. This responsemay occur in a tissue, system, animal or human and includes alleviationof the symptoms of the disease being treated. As used herein withrespect to pseudoviral vectors of the invention, the term“therapeutically effective amount/dose” refers to the amount/dose of avector or pharmaceutical composition containing the vector that issufficient to produce an effective anti-tumor response uponadministration to a subject.

The term “nucleic acids”, as used herein, may be DNA or RNA. Nucleicacids may also include modified nucleotides that permit correct readthrough by a polymerase and do not alter expression of a polypeptideencoded by that nucleic acid. The terms “nucleic acid” and“oligonucleotide” are used interchangeably to refer to a moleculecomprising multiple nucleotides. As used herein, the terms refer tooligoribonucleotides as well as oligodeoxyribonucleotides. The termsshall also include polynucleosides (i.e., a polynucleotide minus thephosphate) and any other organic base containing polymer. Nucleic acidsinclude vectors, e.g., plasmids, as well as oligonucleotides. Nucleicacid molecules can be obtained from existing nucleic acid sources, butare preferably synthetic (e.g., produced by oligonucleotide synthesis).

The phrase “nucleotide sequence” includes both the sense and antisensestrands as either individual single strands or in the duplex.

The phrase “nucleic acid sequence encoding” refers to a nucleic acidwhich directs the expression of a specific protein or peptide. Thenucleic acid sequences include both the DNA strand sequence that istranscribed into RNA and the RNA sequence that is translated intoprotein. The nucleic acid sequences include both the full length nucleicacid sequences as well as non-full length sequences derived from thefull length sequences. It being further understood that the sequenceincludes the degenerate codons of the native sequence or sequences whichmay be introduced to provide codon preference in a specific host cell.

By “DNA” is meant a polymeric form of deoxyribonucleotides (adenine,guanine, thymine, or cytosine) in double-stranded or single-strandedform, either relaxed and supercoiled. This term refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includes single- anddouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having the sequence homologous to the mRNA). Theterm captures molecules that include the four bases adenine, guanine,thymine, or cytosine, as well as molecules that include base analogueswhich are known in the art.

A “gene” or “coding sequence” or a sequence, which “encodes” aparticular protein, is a nucleic acid molecule which is transcribed (inthe case of DNA) and translated (in the case of mRNA) into a polypeptidein vitro or in vivo when placed under the control of appropriateregulatory or control sequences. The boundaries of the gene aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A gene can include, but is notlimited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNAsequences from prokaryotic or eukaryotic DNA, and even synthetic DNAsequences. A transcription termination sequence will usually be located3′ to the gene sequence.

The term “control elements” refers collectively to promoter regions,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (“IRES”), enhancers, and the like, which collectively provide forthe replication, transcription and translation of a coding sequence in arecipient cell. Not all of these control elements need always be presentso long as the selected coding sequence is capable of being replicated,transcribed and translated in an appropriate host cell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

The term “operably linked” refers to an arrangement of elements, whereinthe components so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “5′,” or “3′” relative toanother sequence, it is to be understood that it is the position of thesequences in the non-transcribed strand of a DNA molecule that is beingreferred to as is conventional in the art.

The term “homology” refers to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown in the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions, which form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s), and size determination of thedigested fragments. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when at least about 80%,preferably at least about 90%, and most preferably at least about 95% ofthe nucleotides or amino acids match over a defined length of themolecules, as determined using the methods above.

By “isolated” when referring to a nucleotide sequence, is meant that theindicated molecule is present in the substantial absence of otherbiological macromolecules of the same type. Thus, an “isolated nucleicacid molecule, which encodes a particular polypeptide,” refers to anucleic acid molecule, which is substantially free of other nucleic acidmolecules that do not encode the subject polypeptide; however, themolecule may include some additional bases or moieties, which do notdeleteriously affect the basic characteristics of the composition.

The terms “vector”, “cloning vector”, “expression vector”, and “helpervector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreigngene) can be introduced into a host cell, so as to promote expression(e.g., transcription and/or translation) of the introduced sequence.Vectors include plasmids, phages, viruses, pseudoviruses, etc. As usedherein with respect to the pseudoviral vectors, the term “expressionvector” is used most commonly to refer to a vector that is capable ofinfecting a host cell, while the term “helper vector” is used to referto a vector that is able to mediate proper packaging of the “expressionvector” into a virus-like particle.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting foreign DNA into host cells.

As used herein, the term “transfection” is understood to include anymeans, such as, but not limited to, adsorption, microinjection,electroporation, lipofection and the like for introducing an exogenousnucleic acid molecule into a host cell. The term “transfected” or“transformed”, when used to describe a cell, means a cell containing anexogenously introduced nucleic acid molecule and/or a cell whose geneticcomposition has been altered by the introduction of an exogenous nucleicacid molecule.

As used herein, the term “tumor” refers to a tissue comprisingtransformed cells that grow uncontrollably. A tumor may be benign(benign tumor) or malignant (malignant tumor or cancer). Tumors includeleukemias, lymphomas, myelomas, plasmacytomas, and the like; and solidtumors. Examples of solid tumors that can be treated according to theinvention include sarcomas and carcinomas such as, but not limited to:fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangio sarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat glandcarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, neuroglioma, and retinoblastoma.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviations,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” or“pharmacologically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspension, solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared.

As used herein, the term “heterologous sequence or gene” means a nucleicacid (RNA or DNA) sequence, which is not naturally found in associationwith the nucleic acid sequences of the specified molecule, e.g., apapillomavirus genome. The section below provides greater detail on someapproaches that can be used to prepare virus-like particles andpseudoviruses.

Virus-Like Particles and Pseudovirus Preparation

The term “virus-like particle” (“VLP”) refers to an organized structurecomprising self-assembling ordered arrays of one or more viral capsidproteins that do not include a viral genome. For example, VLPs havingpapillomavirus L1 capsid protein alone, or having both L1 and L2 capsidproteins together can be prepared. The methods used to preparerecombinant capsid particles for many papillomaviruses are known in theart. Some approaches are described, for example, in U.S. PatentPublication No. 2006/0269954, which is hereby expressly incorporated byreference in its entirety.

The term “recombinant protein” refers to a protein that is producedusing molecular biology techniques, for example, recombinant DNAtechnology. As an example, “recombinant protein” can refer to a proteinfrom a genetically engineered nucleic acid, such as a “recombinantnucleic acid construct.” Any protein, peptide, or polypeptide can beencoded by an engineered nucleic acid construct or recombinant nucleicacid construct. The term “protein expression” refers to the processes oftranscription and translation of nucleic acids to produce polypeptides.

“Pseudoviruses” or “papilloma pseudoviruses” or “papillomavirus genetransfer vectors” refer to one or more papillomavirus capsid proteinsthat assemble and package heterologous nucleic acids (e.g., DNA) with orwithout viral nucleic acids (e.g., DNA) into infectious particles. Themethods used to produce papilloma pseudoviruses are known in the art andare described, for example, in U.S. Pat. Nos. 6,599,739, 7,205,126, and6,416,945; and in Buck and Thompson, Production of Papillomavirus-BasedGene Transfer Vectors. Current Protocols in Cell Biology 26.1.1-26.1.19,December 2007, all of which are hereby expressly incorporated byreference in their entireties.

The term “capsomeric structure” or “capsid” or “capsid particle”includes VLPs and pseudoviruses. The following section describes some ofthe diagnostic embodiments contemplated.

Diagnostics

Some embodiments disclosed herein relate to methods for detecting thepresence of cancer cells bound to papilloma pseudovirus or papillomaVLP. Some approaches involve identifying a subject having or suspectedof having cancer cells, administering to the subject a detectable amountof a papilloma pseudovirus or VLP that comprises a detectable label, anddetecting the presence of cancer cells bound to a papilloma pseudovirusor VLP that comprises a detectable label.

Other embodiments disclosed herein relate to methods for detecting thepresence of pre-malignant conditions (e.g., dysplasia orhyperproliferative disease). Some approaches involve identifying asubject having or suspected of having a pre-malignant condition,administering to the subject a detectable amount of a papillomapseudovirus or VLP that comprises a detectable label, and detecting thepresence of pre-malignant cells bound to a papilloma pseudovirus or VLPthat comprises a detectable label.

Embodiments disclosed herein relate to methods to identify all kinds ofcancers, tumors, metastases, and pre-malignant conditions (e.g.,dysplasia or hyperproliferative disease). While not being bound to anyparticular theory, it is believed that the papilloma pseudovirus or VLPselectively binds to and delivers the label to cancer cells withoutbinding to normal cells in intact tissues, where the number of normalcells in intact tissues bound to the pseudovirus or VLP are less than orequal to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4, 0.3%, 0.2%,0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01%of the total number of cells bound by the pseudovirus or VLP.

The detectable label can be a reporter gene carried within the papillomapseudovirus or a label chemically coupled to a capsid protein of thepapilloma pseudovirus or VLP.

Reporter Genes

Since papilloma pseudoviral vectors are gene transfer vectors, it iscontemplated that the cancer cells can be selectively labeled withreporter genes that are incorporated in the pseudovirus. As used hereina “reporter” or a “reporter gene” refers to a nucleic acid moleculecapable of being transcribed as mRNA when operatively linked to apromoter, except that the term “reporter gene” as used herein, is notintended to include wild-type papillomavirus sequences. Preferredreporter genes include luciferase (e.g., firefly luciferase or Renillaluciferase), β-galactosidase, chloramphenicol acetyl transferase (CAT),thymidine kinase (TK), and fluorescent proteins (e.g., green fluorescentprotein, red fluorescent protein, yellow fluorescent protein, bluefluorescent protein, cyan fluorescent protein, or variants thereof,including enhanced variants).

These genes can be incorporated into papilloma pseudoviruses usingtechniques well known to those of ordinary skill in the art. Suitablemethods are described, for example, in Buck and Thompson, Production ofPapillomavirus-Based Gene Transfer Vectors. Current Protocols in CellBiology 26.1.1-26.1.19, December 2007, which is hereby expresslyincorporated by reference in its entirety.

Any reporter nucleic acid sequence may be used as a reporter gene if isit is detectable by a reporter assay. Reporter assays include any knownmethod for detecting a nucleic acid sequence or its encoded proteinproduct directly or indirectly. Reporter assays can be conducted invitro or in vivo. For example, a reporter assay can measure the level ofreporter gene expression or activity by measuring the level of reportermRNA, the level of reporter protein, or the amount of reporter proteinactivity. The level of reporter mRNA may be measured, for example, usingRT-PCR, ethidium bromide staining of a standard RNA gel, Northernblotting, primer extension, or nuclease protection assay. The level ofreporter protein may be measured, for example, using chemiluminescence,microscopic analysis, Coomassie staining of an SDS-PAGE gel, Westernblotting, dot blotting, slot blotting, ELISA, or RIA. Reporter proteinactivity may be measured using an assay specific to the reporter beingused. For example, standard assays for luciferase, CAT, β-galactosidase,thymidine kinase (TK) assays (including full body scans; see Yu, Y. etal. (2000) Nature Medicine 6:933-937 and Blasberg, R. (2002) J. Cereb.Blood Flow Metab. 22:1157-1164), and fluorescent proteins are allwell-known in the art. For instance, a Maestro (CRi, Woburn, Mass.)imaging device can be used to detect reporter gene expression.

Presence of the label can be detected in the subject using methods knownin the art for in vivo scanning. These methods depend upon the type oflabel used. Skilled artisans are able to determine the appropriatemethod for detecting a particular label. Methods and devices that may beused in the diagnostic methods of the invention include, but are notlimited to, computed tomography (CT), whole body scan such as positionemission tomography (PET), single photon emission tomography (SPECT),magnetic resonance imaging (MRI), sonography, chemiluminescence, and theMaestro™ in-vivo imaging system (CRi, Inc.). In vivo scanning can beconducted in a local region of the subject (for example, the esophagealarea can be scanned) or whole body scanning can be conducted.

Cancer cells expressing the genetic markers delivered by pseudovirusescan be identified as follows: for the HSV-tk gene, the subject can beadministered radiolabeled 9-[(4[¹⁸F]fluro-3-hydroxymethylbutyl)guanine(FHBG), administered intravenously, about 6000 μCi/Kg body weight of therecipient, (commercially available from PET Imaging Science Center, U.of South California). Expression of HSV-tk activity in cancer cellsresults in the accumulation of radiolabeled FHBG and can be monitored byPositron Emission Tomography (PET). In vivo GFP expressing cancer cellscan be monitored by fluorescence microscopic examination of tissuesections. Tissue sections of Flue or Rluc expressing cancer cells can bemonitored by Cooled Charge-Coupled Device (CCD) cameras in vivo(commercially available from Xenogen Corp., Alameda, Calif.). D₂Ractivity can be identified by administering3-(2-¹⁸F]fluoroethyl)spiperone ([¹⁸F]FESP) and monitored by PET. Thefollowing section describes examples of labels which can be chemicallycoupled to pseudoviruses or VLPs and examples of methods that can beused to chemically couple labels to pseudoviruses or VLPs.

Chemically Coupled Labels

Some embodiments also relate to methods of identifying cancers, tumors,metastases and pre-malignant conditions using papilloma pseudoviruses orVLPs labeled via chemical coupling. Chemically coupled labels include,but are not limited to, fluorescent dyes, phosphors, radionuclides, andother molecules known in the art that can be detected directly orindirectly.

Examples of fluorescent dyes include, but are not limited to,7-Amino-actinomycin D, Acridine orange, Acridine yellow, Alexa Fluordyes (Molecular Probes), Auramine O, Auramine-rhodamine stain,Benzanthrone, 9,10-Bis(phenylethynyl)anthracene,5,12-Bis(phenylethynyl)naphthacene, CFDA-SE, CFSE, Calcein,Carboxyfluorescein, 1-Chloro-9,10-bis(phenylethynyl)anthracene,2-Chloro-9,10-bis(phenylethynyl)anthracene, Coumarin, Cyanine, DAPI,Dark quencher, Dioc6, DyLight Fluor dyes (Thermo Fisher Scientific),Ethidium bromide, Fluorescein, Fura-2, Fura-2-acetoxymethyl ester, Greenfluorescent protein and derivatives, Hilyte Fluor dyes (AnaSpec),Hoechst stain, Indian yellow, Luciferin, Perylene, Phycobilin,Phycoerythrin, Phycoerythrobilin, Propidium iodide, Pyranine, Rhodamine,RiboGreen, Rubrene, Ruthenium(II) tris(bathophenanthroline disulfonate),SYBR Green, Stilbene, Sulforhodamine 101, TSQ, Texas Red, Umbelliferone,or Yellow fluorescent protein.

Examples of phsosphors include, but are not limited to Phosphor,Anthracene, Barium fluoride, Bismuth germanate, Cadmium sulfide, Cadmiumtungstate, Gadolinium oxysulfide, Lanthanum bromide, Polyvinyl toluene,Scheelite, Sodium iodide, Stilbene, Strontium aluminate, Yttriumaluminium garnet, Zinc selenide, or Zinc sulfide.

Examples of suitable radioisotopic labels include, but are not limitedto, ³H, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu,⁹⁰Y, ⁶⁷Cu, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ¹⁸⁶Re, ¹⁸⁸Re, or ²¹²Bi. Preferableradiolabeled pseudoviruses or VLPs are able to deliver more than 6000rads to the tumor and have sufficient affinity so that the patient'sbone marrow is not exposed to more than 300 rads. In some embodiments,100, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000,6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10000 rads to the cancercells For example, ¹³¹I labeled coupled to the surface of pseudovirusesor VLPs is one example of a radiolabeled pseudoviruses or VLPs withinthe scope of these embodiments. Use of ¹³¹I labeled pseudoviruses orVLPs as well as other radiolabeled pseudoviruses or VLPs, is also withinthe scope of these embodiments. The pseudoviruses or VLPs can beradiolabeled, for example, by the Iodogen method according toestablished methods.

The detection may occur in vitro or in vivo. For example, fluorescentdyes (e.g., Alexa Fluor 488) can be coupled to the pseudoviruses bymethods well known in the art (see, for example, Buck and Thompson,Production of Papillomavirus-Based Gene Transfer Vectors. CurrentProtocols in Cell Biology 26.1.1-26.1.19, December 2007, which is herebyexpressly incorporated by reference in its entirety).

In some embodiments, a radioactive imaging compound is chemicallycoupled to the pseudoviruses or VLPs. Radioactive chemical tracers whichemit radiation such as gamma rays can be coupled to the pseudoviruses orVLPs to provide diagnostic information. In the case of yttrium oxideencasing layers, a positron emitter such as ⁸⁷Y can be added to allowimaging. In the case of lanthanum phosphate, there are a variety ofgamma emitters that may be used to add an imaging component to thetreatment component.

A label may be chemically coupled directly to the pseudovirus or VLP(e.g., without a linking group) through an amino group, a sulfhydrylgroup, a hydroxyl group, or a carboxyl group.

In some embodiments, a label is attached to the pseudovirus or VLP via alinking group. The linking group can be any biocompatible linking group,where “biocompatible” indicates that the compound or group can benon-toxic and may be utilized in vitro or in vivo without causinginjury, sickness, disease, or death. The label can be bonded to thelinking group, for example, via an ether bond, an ester bond, a thiolbond or an amide bond. Suitable biocompatible linking groups include,but are not limited to, an ester group, an amide group, an imide group,a carbamate group, a carboxyl group, a hydroxyl group, a carbohydrate, asuccinimide group (including, for example, succinimidyl succinate (SS),succinimidyl propionate (SPA), succinimidyl butanoate (SBA),succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA) orN-hydroxy succinimide (NHS)), an epoxide group, an oxycarbonylimidazolegroup (including, for example, carbonyldimidazole (CDI)), a nitro phenylgroup (including, for example, nitrophenyl carbonate (NPC) ortrichlorophenyl carbonate (TPC)), a trysylate group, an aldehyde group,an isocyanate group, a vinylsulfone group, a tyrosine group, a cysteinegroup, a histidine group or a primary amine.

Chemically coupled labels can be detected using any of the methodsdescribed for detecting reporter genes. In one embodiment, the papillomapseudovirus or VLP is labeled with a radioisotope and is detected in thepatient using a radiation responsive surgical instrument (Thurston etal., U.S. Pat. No. 5,441,050). In another embodiment, the papillomapseudovirus or VLP is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the papilloma pseudovirus or VLP islabeled with a positron emitting metal and is detected in the patentusing positron emission-tomography. In yet another embodiment, thepapilloma pseudovirus or VLP is labeled with a paramagnetic label and isdetected in a patient using magnetic resonance imaging (MRI). Thefollowing section provides greater detail on some embodiments that canbe used to monitor cancer therapy.

Monitoring Cancer Therapy

The phrase “monitoring cancer therapy” refers to determining therelative amount of cancer cells in the body of a patient before, duringand/or after anti-cancer therapy.

Some embodiments disclosed herein relate to methods for monitoring theprogress or efficacy of cancer therapy in a subject. Subjects identifiedas having cancer and undergoing cancer therapy can be administeredpapillomavirus pseudovirus or VLP including labels as described above.

Subjects can be administered a papilloma pseudovirus or VLP thatincludes a label before the onset of treatment or during treatment.Cells containing the label can be assayed for and this measurement canbe compared to one obtained at a subsequent time during the therapyand/or after therapy has been completed. In this way, it is possible toevaluate the inhibition of cancer cell proliferation, and theeffectiveness of the therapy. Since only living cancer cells willcontain the label, the therapy can continue until a minimal amount oflabel is detected.

Some embodiments disclosed herein also relate to methods for determiningthe amount of cancer cells present in a subject. By detecting the label,one can determine whether cancer cells are present within the subjectand the amount of label measured is proportional to the amount of cancercells present in the subject.

Diagnostic and Therapeutic Kits

Some embodiments include methods that utilize the pseudoviruses or VLPsin kits for the detection and/or treatment of tumors. The kits are basedon the pseudovirus' or VLP's enhanced specificity towards cancer cellsrather than a non-cancerous cells.

The diagnostic kits can comprise an effective amount of a labeledpapilloma pseudovirus or VLP. The kits can further comprise anappropriate amount of non-cancerous control cells. The pseudovirus, VLPand/or cells may be supplied either frozen, lyophilized or growing onsolid or in liquid medium. The diagnostic kits can further compriseinert ingredients and other kit components such as vials, applicators,packaging components and the like, which are known to those skilled inthe art.

In an embodiment, a kit for the diagnostic detection of cervical cancercan be assembled. The kit can include a papilloma pseudovirus or VLPincluding a label (for example, a fluorescent label). The pseudovirus orVLP can be present in the kit in a liquid medium which can be aspiratedonto the cervicovaginal mucosa of a subject. After an incubation periodto allow the pseudovirus or VLP to selectively attach to suspectedcancer cells, the cervicovaginal mucosa can be washed to remove excessunbound pseudovirus or VLP. Subsequently a detection device (forexample, a fluorescent detection device) can be used to detect and/ormeasure the label included in the pseudovirus or VLP. The detection oflabel will indicate the presence of cancer cells.

Biomedical Applications

Embodiments disclosed herein also relate to methods of selectivelyinhibiting the proliferation of cancer cells (or pre-malignant cells)and/or killing cancer cells (or pre-malignant cells) without inhibitingproliferation of and/or killing normal cells. In some approaches, asubject that has cancer is identified using clinical or diagnostictechniques known in the art. The subject is then provided an inhibitoryamount of papilloma pseudovirus or VLP that includes a therapeuticagent. Because the papilloma pseudovirus or VLP selectively attaches tocancer cells, a very focused and sensitive cancer therapy can beprovided. In some embodiments, a pre-malignant condition can be treatedusing methods disclosed herein.

In some contexts, The phrase “selectively inhibiting” or “specificinhibition” indicates that the amount of normal cells that exhibit aninhibition of proliferation or are killed is less than or equal to 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4, 0.3%, 0.2%, 0.1%, 0.09%,0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.001%, 0.0001%,0.00001%, or 0% of the total number of cells that have been contactedwith the papilloma pseudovirus or VLP or at an inoculation site (e.g., asite of 1 cm², 1 mm², 1 μm², or 1 nm²). A determination of specificinhibition, specific binding, or selective inhibition or selectivebinding of pseudoviruses or VLPs to cancer cells or pre-malignant cellscan be determined by a range of methods known in the art or as describedherein (e.g., competitive binding assays or Scatchard analyses). In somecontexts, specific binding, specific inhibition, or selective binding orselective inhibition can be determined by mere observation, as shown inExample 11.

Therapeutic Genes

Therapeutic agents include, but are not limited to, therapeutic genes,proteins encoded by therapeutic genes, cytotoxins, and radionuclides.Therapeutic genes include, but are not limited to, tumor suppressorgenes, pro-apoptotic genes, cytokines, enzymes, hormones, andimmunomodulatory genes.

A “therapeutic gene” refers to a gene which can be administered to asubject for the purpose of treating or preventing a disease. Forexample, a therapeutic gene can be a gene administered to a subject fortreatment of cancer. Examples of therapeutic genes include, but are notlimited to, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zac1,scFV ras, DCC, NF-1, NF-2, WT-I, MEN-I, MEN-II, BRCA I, VHL, MMAC1, FCC,MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11 IL-12, GM-CSF, G-CSF, thymidine kinase, mda7, fus, interferonalpha, interferon beta, interferon gamma, ADP, p53, ABLI, BLC1, BLC6,CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS2, ETV6, FGR, FOX, FYN,HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS,P1M1, PML, RET, SRC, TAL1, TCL3, YES, MADH4, RB1, TP53, WT1, TNF, BDNF,CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NTS, ApoAI, ApoAIV, ApoE, RaplA,cytosine deaminase, Fab, ScFv, BRCA2, zac1, ATM, HIC-1, DPC-4, FHIT,PTEN, ING1, NOEY1, NOEY2, OVCA1, MADR2, 53BP2, IRF-1, Rb, zac1, DBCCR-1,rks-3, COX-1, TFPI, PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk,ret, gsp, hst, abl, E1A, p300, VEGF, FGF, thrombospondin, BAI-1, GDAIF,or MCC.

In certain embodiments, the therapeutic gene can be a tumor suppressorgene. A tumor suppressor gene refers to a gene that, when present in acell, reduces the tumorigenicity, malignancy, or hyperproliferativephenotype of the cell. This definition includes both the full lengthnucleic acid sequence of the tumor suppressor gene, as well as non-fulllength sequences of any length derived from the full length sequences.It being further understood that the sequence includes the degeneratecodons of the native sequence or sequences which may be introduced toprovide codon preference in a specific host cell.

Examples of tumor suppressor nucleic acids within this definitioninclude, but are not limited to, APC, CYLD, HIN-1, KRAS2b, p16, p19,p21, p27, p27mt, p53, p57, p73, PTEN, Rb, Uteroglobin, Skp2, BRCA-1,BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2/JV18, MEN1, MEN2, MTS1, NF1, NF2,VHL, WRN, WT1, CFTR, C-CAM, CTS-1, zac1, scFV, MMAC1, FCC, MCC, Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), 101F6, Gene 21 (NPRL2), or a gene encoding a SEM A3polypeptide and FUS1. Other exemplary tumor suppressor genes aredescribed in publicly available databases of tumor suppressor genes.Nucleic acids encoding tumor suppressor genes, as discussed above,include tumor suppressor genes, or nucleic acids derived therefrom(e.g., cDNAs, cRNAs, mRNAs, and subsequences thereof encoding activefragments of the respective tumor suppressor amino acid sequences), aswell as vectors comprising these sequences. One of ordinary skill in theart would be familiar with tumor suppressor genes that can be applied inthe embodiments.

In certain embodiments, the therapeutic gene can be a gene that inducesapoptosis (i.e., a pro-apoptotic gene). A “pro-apoptotic gene amino acidsequence” refers to a polypeptide that, when present in a cell, inducesor promotes apoptosis. The present invention contemplates inclusion ofany pro-apoptotic gene known to those of ordinary skill in the artExemplary pro-apoptotic genes include CD95, caspase-3, Bax, Bag-1,CRADD, TSSC3, bax, hid, Bak, MKP-7, PERP, bad, bcl-2, MST1, bbc3, Sax,BIK, BID, and mda7. One of ordinary skill in the art would be familiarwith pro-apoptotic genes, and other such genes not specifically setforth herein that can be applied in the methods and compositions of thepresent invention.

The therapeutic gene can also be a gene encoding a cytokine. The term‘cytokine’ is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators. A“cytokine” refers to a polypeptide that, when present in a cell,maintains some or all of the function of a cytokine. This definitionincludes full-length as well as non-full length sequences of any lengthderived from the full length sequences. It being further understood, asdiscussed above, that the sequence includes the degenerate codons of thenative sequence or sequences which may be introduced to provide codonpreference in a specific host cell.

Examples of such cytokines include, but are not limited to, lymphokines,monokines, growth factors and traditional polypeptide hormones. Includedamong the cytokines are growth hormones such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; prostaglandin, fibroblast growth factor; prolactin;placental lactogen, OB protein; tumor necrosis factor-alpha and -beta:mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-beta;platelet-growth factor; transforming growth factors (TGFs) such asTGF-alpha and TGF-beta; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs)such as macrophage-C SP (M-CSF); granulocyte-macrophage-CSF (GM-CSF);and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 IL-11,IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-24,LIF, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand or FLT-3.

Other examples of therapeutic genes include genes encoding enzymes.Examples include, but are not limited to, ACP desaturase, an ACPhydroxylase, an ADP-glucose pyrophorylase, an ATPase, an alcoholdehydrogenase, an amylase, an amyloglucosidase, a catalase, a cellulase,a cyclooxygenase, a decarboxylase, a dextrinase, an esterase, a DNApolymerase, an RNA polymerase, a hyaluron synthase, a galactosidase, aglucanase, a glucose oxidase, a GTPase, a helicase, a hemicellulase, ahyaluronidase, an integrase, an invertase, an isomerase, a kinase, alactase, a lipase, a lipoxygenase, a lyase, a lysozyme, apectinesterase, a peroxidase, a phosphatase, a phospholipase, aphosphorylase, a polygalacturonase, a proteinase, a peptidease, apullanase, a recombinase, a reverse transcriptase, a topoisomerase, axylanase, a reporter gene, an interleukin, or a cytokine.

Further examples of therapeutic genes include the gene encodingcarbamoyl synthetase I, ornithine transcarbamylase, arginosuccinatesynthetase, arginosuccinate lyase, arginase, fumarylacetoacetatehydrolase, phenylalanine hydroxylase, alpha-antitrypsin,glucose-6-phosphatase, low-density-lipoprotein receptor, porphobilinogendeaminase, factor VIII, factor IX, cystathione beta.-synthase, branchedchain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase,propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoAdehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, hepaticphosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein,T-protein, Menkes disease copper-transporting ATPase, Wilson's diseasecopper-transporting ATPase, cytosine deaminase, hypoxanthine-guaninephosphoribosyltransferase, galactose-1-phosphate uridyltransferase,phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase,alpha-L-iduronidase, glucose-6-phosphate dehydrogenase, HSY thymidinekinase, or human thymidine kinase.

Therapeutic genes also include genes encoding hormones. Examplesinclude, but are not limited to, genes encoding growth hormone,prolactin, placental lactogen, luteinizing hormone, follicle-stimulatinghormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin,adrenocorticotropin, angiotensin I, angiotensin II, beta-endorphin,beta-melanocyte stimulating hormone, cholecystokinin, endothelin I,galanin, gastric inhibitory peptide, glucagon, insulin, lipotropins,neurophysins, somatostatin, calcitonin, calcitonin gene related peptide,beta-calcitonin gene related peptide, hypercalcemia of malignancyfactor, parathyroid hormone-related protein, parathyroid hormone-relatedprotein, glucagon-like peptide, pancreastatin, pancreatic peptide,peptide YY, PHM, secretin, vasoactive intestinal peptide, oxytocin,vasopressin, vasotocin, enkephalinamide, metorphinamide, alphamelanocyte stimulating hormone, atrial natriuretic factor, amylin,amyloid P component, corticotropin releasing hormone, growth hormonereleasing factor, luteinizing hormone-releasing hormone, neuropeptide Y,substance K, substance P, or thyrotropin releasing hormone.

An “immunostimulatory nucleic acid or gene” as used herein is anynucleic acid containing an immunostimulatory motif or backbone thatinduces an immune response. The immune response may be characterized as,but is not limited to, a Th1-type immune response or a Th2-type immuneresponse. Such immune responses are defined by cytokine and antibodyproduction profiles which are elicited by the activated immune cells.

Examples of the immunomodulatory genes include chemokines, adhesivemolecules, cytokines, co-stimulatory molecule, growth factors, andreceptor molecules. The chemokines include MIP-1 alpha, MIP-1 beta,RANTEs, IL-8 and MCP-1. Examples of the adhesive molecules includeselectin family constructs, mucin-like molecules, integrin familyconstructs, and immunoglobulin superfamily constructs. Examples of theselect in family constructs include L-selectin, P-selectin, andE-selectin. The mucin-like molecules are ligands for the selectin familyconstructs. Examples of the mucin-like molecule include CD34, GlyCAM-1,and MadCAM-1. Examples of the integrin family constructs include LFA-1,VLA-1, Mac-1, and p150.95. Examples of the immunoglobulin superfamilyconstructs include PECAM-1, ICAMs (ICAM-1, ICAM-2, and ICAM-3), CD2, andLFA-3. Examples of the cytokine include mutants of M-CSF, GM-CSF, G-CSF,CSF, IL-4, and IL-18 (including deletion of the first about 35 aminoacid residues which are present in the precursor of a protein but arenot present in the protein in the mature form). Examples ofco-stimulatory molecules include B71, B72, CD40 and CD40 ligands(CD40L). Examples of growth factors include IL-7, nerve growth factors,and a vascular endothelial growth factor. Examples of the receptormolecules include a Fas lethal gene expression product, a tumor necrosisfactor TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR,LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, and DR6. Thecompositions of the present invention may contain caspase (ICE).

Therapeutic genes also include genes encoding polypeptides which arecytotoxic to cancer cells. Cytotoxic proteins include, but are notlimited to, ricin, pokeweed toxin, diphtheria toxin A, saporin, gelonin,and Pseudomonas exotoxin A.

As will be understood by those in the art, the term “therapeutic gene”includes genomic sequences, cDNA sequences, and smaller engineered genesegments that express, or may be adapted to express, proteins,polypeptides, domains, peptides, fusion proteins, and mutants. Thenucleic acid molecule encoding a therapeutic gene may comprise acontiguous nucleic acid sequence of about 5 to about 12000 or morenucleotides, nucleosides, or base pairs.

Chemically Coupled Therapeutic Agents

Embodiments disclosed herein also relate to methods of inhibiting theproliferation of cancers, tumors, and metastases using papillomapseudoviruses or VLPs chemically coupled to therapeutic agents.Chemically coupled therapeutic agents include, but are not limited to,therapeutic proteins as described above, cytotoxins, and radionuclides.

Cytotoxins include, but are not limited to, ricin, pokeweed toxin,diphtheria toxin A, saporin, gelonin, and Pseudomonas exotoxin A.

In an embodiment papilloma pseudoviruses or VLPs can be coupled to aradionuclide particle. The pseudovirus or VLP can attach to a bindingsite on the target cancer cells, and the radionuclide can administer alethal dose of radiation. The basic strategy of radionuclide treatmentis that coupling of a radionuclide to the pseudovirus or VLP causesenhanced accumulation of the radionuclide at the targeted site.Accumulation of the radionuclide at the targeted site causes radiationtherapy to be delivered near the targeted site with a radiusapproximating the mean path length of the emitted particle.

Several different radionuclides can be considered for therapy. Thechoice of radionuclide takes into account the physical and chemicalcharacteristics of the radionuclide, including half-life, radiationemission properties, radiolabeling properties, availability, in vivodistribution and stability. Suitable radionuclides possess a half-lifelong enough for target localization, little or no gamma radiation,intermediate beta particle energy, stable daughter products, and stablefixation with an antibody system. Many β-particle-emitting radionuclidesare available. These include, for example, yttrium-90 (⁹⁰Y), iodine-131(¹³¹I), copper-67 (⁶⁷Cu) and rhenium-186 (⁸⁶Re) Alpha (α)particle-emitting radionuclides include astatine-211 (²¹¹At) andbismuth-212 (²¹²Bi). Alpha and beta emitters are preferred because themean path links are limited to dozens of mm, thereby limiting treatmentto the immediate vicinity of the target. Beta particles may be moresuitable for larger tumors due to the longer mean path length of thebeta emission. Alpha particles generally have extremely high energies(greater than 5 MeV) and high linear energy transfer rates, which areuseful for delivering high doses to a limited area.

Further embodiments disclosed herein relate to combinations ofdiagnostic and/or therapeutic methods described herein. For example,pseudoviruses can be constructed that comprise a therapeutic gene and aradionuclide. In other embodiments, pseudoviruses can be constructedthat comprise Oligo T RNA and therapeutic gene.

Prodrugs

The term “prodrug” as used herein refers to a drug which is inactive asit is and becomes active when it is chemically changed in the body by adrug-metabolizing enzyme (e.g., purine and pyrimidine derivatives usedas chemotherapeutic agents for cancer). Examples of the prodrugs hereinpreferably include ganciclovir, acyclovir, taxol, camptothecin, guaninenucleoside derivatives (e.g., A-5021), and the like. A prodrug hereinpreferable for the present invention is a prodrug which is converted toan active form by a suicide gene contained in a papilloma pseudovirus orVLP.

The term “suicide gene” as used herein refers to a gene which can killthe cell in which it is expressed. Representatively, such a gene is ametabolically toxic gene. For example, a method for introducing asuicide gene incorporated into a pseudovirus or VLP construct intocancer cells to drive them to suicide is herein exemplified. Forexample, thymidine kinase may be incorporated into a pseudovirus or VLP.

Oligo T RNA to Induce Tumor Regression

Anti-tumor therapeutic vaccines and anti-tumor cytotoxic gene therapyhave produced limited clinical success, despite extensive effort. Asimple approach that could combine the two activities might lead to moreeffective anti-tumor therapy. To accomplish this, a gene transfer vectorthat expresses oligo T RNA was constructed. In some embodiments, the RNAis expressed from a promoter, e.g. a Pol III promoter, as part of apapillomavirus pseudogenome after PsV transduction. This RNA will not bepolyadenylated but will form a duplex with the poly A tails of cellularmRNAs. The double strand RNAs thus generated can lead to cytotoxicity byactivation of PKR-mediated apoptosis and immunity through activation ofTLR 3. The small size and dual function of this expression cassetteleave open the possibility of expressing other genes in the up to 8 kbpseudogenome. Genes to increase immunogenicity, such as GMCSF, orcytotoxicity, such as TK, could be cotransduced by the oligo T PsV.Oligo T PsVs cannot be efficiently produced in most cells since theoligo T would be generated in the PsV producer cells and thus induceapoptosis prior to PsV assembly. However 293, and 293-derived lines,express adenovirus VA RNAs, which interact with PKR and prevent itsactivation by dsRNA. Oligo T PsV can be efficiently produced in a 293TTline. Other suitable cell lines include those that can express VA 1 RNAsand SV40 Large T-antigen such as 293FT cells (Invitrogen). The oligo Tpseudogenome induced cytotoxicity after introduction into epitheliallines lacking VA RNAs. The Oligo T nucleic acid can be less than orequal to 200, 175, 150, 125, 100, 95, 80, 75, 70, 65, 60, 55, 50, 45,40, 35, 30, 25, 20, 15, or 10 nucleotides.

Formulations

Embodiments disclosed herein also relate to methods of administeringpseudoviruses or VLPs to a subject in order to contact cancer cells withpseudoviruses or VLPs. The routes of administration can vary with thelocation and nature of the tumor, and include, e.g., intravascular,intradermal, transdermal, parenteral, intravenous, intramuscular,intranasal, subcutaneous, regional, percutaneous, intratracheal,intraperitoneal, intraarterial, intravesical, intratumoral, inhalation,perfusion, lavage, direct injection, and oral administration andformulation.

The term “intravascular” is understood to refer to delivery into thevasculature of a patient, meaning into, within, or in a vessel orvessels of the patient. In certain embodiments, the administration canbe into a vessel considered to be a vein (intravenous), while in othersadministration can be into a vessel considered to be an artery. Veinsinclude, but are not limited to, the internal jugular vein, a peripheralvein, a coronary vein, a hepatic vein, the portal vein, great saphenousvein, the pulmonary vein, superior vena cava, inferior vena cava, agastric vein, a splenic vein, inferior mesenteric vein, superiormesenteric vein, cephalic vein, and/or femoral vein. Arteries include,but are not limited to, coronary artery, pulmonary artery, brachialartery, internal carotid artery, aortic arch, femoral artery, peripheralartery, and/or ciliary artery. It is contemplated that delivery may bethrough or to an arteriole or capillary.

Injection into the tumor vasculature is specifically contemplated fordiscrete, solid, accessible tumors. Local, regional or systemicadministration also may be appropriate. For tumors of greater than about4 cm, the volume to be administered can be about 4-10 ml (preferably 10ml), while for tumors of less than about 4 cm, a volume of about 1-3 mlcan be used (preferably 3 ml). Multiple injections delivered as singledose comprise about 0.1 to about 0.5 ml volumes. The pseudoviruses orVLPs may advantageously be contacted by administering multipleinjections to the tumor, spaced at approximately 1 cm intervals.

In the case of surgical intervention, pseudoviruses or VLPs can beadministered preoperatively, to render an inoperable tumor subject toresection. Alternatively, pseudoviruses or VLPs can be administered atthe time of surgery, and/or thereafter, to treat residual or metastaticdisease. For example, a resected tumor bed may be injected or perfusedwith a formulation comprising pseudovirus or VLP that renders thepseudovirus or VLP advantageous for treatment of tumors. The perfusionmay be continued post-resection, for example, by leaving a catheterimplanted at the site of the surgery. Periodic post-surgical treatmentcan be carried out.

Continuous administration also may be applied where appropriate, forexample, where a tumor is excised and the tumor bed is treated toeliminate residual, microscopic disease. Such continuous perfusion maytake place for a period from about 1-2 hours, to about 2-6 hours, toabout 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2wk or longer following the initiation of treatment. Generally, the doseof the therapeutic composition via continuous perfusion will beequivalent to that given by a single or multiple injections, adjustedover a period of time during which the perfusion occurs.

Treatment regimens may vary as well, and often depend on tumor type,tumor location, disease progression, and health and age of the patient.Obviously, certain types of tumor will require more aggressivetreatment, while at the same time, certain patients cannot tolerate moretaxing protocols. The clinician will be best suited to make suchdecisions based on the known efficacy and toxicity (if any) of thetherapeutic formulations.

In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with therapeutic pseudoviralconstructs or VLPs may increase the resectability of the tumor due toshrinkage at the margins or by elimination of certain particularlyinvasive portions. Following treatments, resection may be possible.Additional treatments subsequent to resection can serve to eliminatemicroscopic residual disease at the tumor site.

A typical course of treatment, for a primary tumor or a post-excisiontumor bed, can involve multiple doses. Typical primary tumor treatmentcan involve a 6 dose application over a two-week period. The two-weekregimen may be repeated one, two, three, four, five, six or more times.During a course of treatment, the need to complete the planned dosingsmay be re-evaluated.

The treatments may include various “unit doses.” Unit dose refers to adose containing a predetermined-quantity of the therapeutic composition.The quantity to be administered, and the particular route andformulation, are within the skill of those in the clinical arts. A unitdose need not be administered as a single injection but may comprisecontinuous infusion over a set period of time. Unit dose of the presentinvention may conveniently be described in terms of plaque forming units(pfu) for a viral construct. Unit doses range from 10³, 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ pfu and higher. Alternatively,depending on the kind of pseudovirus and the titer attainable, one willdeliver 1 to 100, 10 to 50, 100-1000, or up to about 10⁴, 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵ or higher infectiouspseudoviral particles to the patient or to the patient's cells.

Injectable Compositions and Formulations

Injection of pseudoviruses or VLPs can be delivered by syringe or anyother method used for injection of a solution, as long as thepseudovirus or VLP can pass through the particular gauge of needlerequired for injection. A novel needleless injection system has recentlybeen described (U.S. Pat. No. 5,846,233) having a nozzle defining anampule chamber for holding the solution and an energy device for pushingthe solution out of the nozzle to the site of delivery. A syringe systemhas also been described for use in gene therapy that permits multipleinjections of predetermined quantities of a solution precisely at anydepth (U.S. Pat. No. 5,846,225).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerolpropylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thirnerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intratumoral and intraperitonealadministration. In this connection, sterile aqueous media that can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug release capsules and the like.

The following examples provide illustrations of some of the embodimentsdescribed herein but are not intended to limit invention.

Example 1 Genital Transmission of HPV in a Mouse Model is Potentiated byNonoxynol-9 and Inhibited by Carrageenan

A mouse model of cervicovaginal infection with HPV16 that recapitulatesthe establishment phase of papillomavirus infection was developed asfollows.

Six- to eight-week-old female BALB/cAnNCr mice were obtained from theNational Institutes of Health and housed and handled in accordance withtheir guidelines. Experimental protocols were approved by the NationalCancer Institute's Animal Care and Use Committee. Unless otherwisenoted, all mice received 3 mg of Depo-Provera (Pfizer) diluted in 100 μlof sterile PBS in a subcutaneous injection 4 d before pseudoviruschallenge.

For vaginal challenge, mice designated for N-9 pretreatment received 50μl of the N-9 containing compound intravaginally 6 h before intravaginalinoculation with pseudovirus. The material was delivered with an M50positive-displacement pipette (Gilson), and standard dissecting forcepswere used to occlude the vaginal introitus to achieve maximal retentionof the material Mice designated for mechanical disruption underwent aprocedure in conjunction with pseudovirus inoculation in which aCytobrush cell collector (Cooper-Surgical) was inserted in the vaginaand twirled clockwise and counterclockwise 10 times. The pseudovirusinoculum was a 20-μl dose composed of 5 μl of purified pseudovirus witha titer of −5×10⁹ IU/ml mixed with 15 μl of a 3% carboxymethylcellulose(CMC) preparation, with the exceptions of certain experiments in which 5μl of inoculum was mixed with 5 μl of the indicated preparation, or inwhich 15 μl of inoculum was mixed with 5 μl of 4% CMC. In theN-9-pretreated mice, this dose was delivered as a one-time, atraumatic,intravaginal inoculation using an M20 positive-displacement pipette. Inthe Cytobrush-treated mice, the inoculum was delivered in two doses, 10μl before and 10 μl after Cytobrush treatment, using an M20positive-displacement pipette. Unless otherwise indicated, thereproductive tract was harvested on day 3 post-challenge after the micewere euthanized by CO2 inhalation. For endocervical challenge, theendocervical canal was pretreated by direct instillation of 15 μl of 1%CMC or 15 μl of 1% CMC with 4% N-9. Six hours after pretreatment, 7 μl(−1.4×10⁷ IU) of pseudovirus mixed with 7 μl of 1% CMC was alsodeposited directly into the endocervical canal.

The results of these initial tests showed that the mouse model ofcervicovaginal infection with HPV16 successfully recapitulated theestablishment phase of papillomavirus infection. In the next example,the effects of nonoxynol-9 and carrageenan in the mouse model wereevaluated

Example 2 Genital Transmission of HPV in a Mouse Model is Potentiated byNonoxynol-9 and Inhibited by Carrageenan

The ability of pseudoviruses to infect mechanically damaged cells,nononoxynol-9 treated cells, and carrageenan treated cells wasinvestigated. The details of these experiments follows.

Gentle mechanical abrasion of the genital epithelium with a Cytobrushcell collector permitted detectable levels of pseudovirus infection.Whether chemical disruption of the genital epithelium could promoteinfection was also determined. N-9 is a nonionic, membrane-activesurfactant that is widely used as a spermicide and is known to disruptthe normal architecture of animal and human genital epithelium. Aformulation of 3% carboxymethylcellulose (CMC) designated to mimic theviscosity of a typical vaginal lubricant gel was made with or without 4%N-9. The gels were instilled in the vagina 6 h before the mice wereinoculated intravaginally with pseudovirus. The mice pretreated with CMCalone were not detectably infected, whereas those pretreated with eitherCytobrush or with CMC and N-9 were highly susceptible to infection(P=0.05, 0.003, respectively) (FIG. 1a ). Indeed, reporter signalintensity in the latter group was an average of fivefold stronger thaninfection-related signal induced by Cytobrush treatment (P=0.008).Conceptrol, an over-the-counter, CMC-based spermicide that contains 4%N-9, also sensitized the genital tract to pseudovirus infection to agreater degree than did Cytobrush treatment (P=0.02).

A wide range of genital HPV types can be potently inhibited in vitro bycarrageenan, an inexpensive polysaccharide whose gelling properties haveled to its incorporation into some over-the-counter vaginal lubricants.To test whether carrageenan can block infectivity in vivo, mice werechallenged with HPV16 pseudoviruses premixed 1:1 with either 1%t-carrageenan or a 3% CMC preparation to control for the viscosity ofthe carrageenan preparation. Carrageenan prevented infection in thegenital mucosa rendered susceptible to infection by either mechanicaldisruption (Cytobrush) or chemical disruption (N-9) (FIG. 1b ). Twocommercial carrageenan-containing lubricants (Divine No. 9 and BIOglide)that showed strong inhibitory activity in an in vitro pseudovirus assaysimilarly prevented detectable infection in vitro (FIG. 1c ). To moreclosely mimic the conditions under which carrageenan might be used incommon practice as a topical microbicide to prevent genital HPVtransmission, N-9 in carrageenan or N-9 in control CMC gel was appliedintravaginally 6 h before pseudovirus challenge. As expected, theCMC-based gel containing N-9 rendered the mucosa susceptible tosignificant HPV pseudovirus infection (P=0.03), while thecarrageenan-based gel prevented detectable infection (FIG. 1d ). Wheneach of the carrageenan conditions were compared to the negativecontrols, P values were >0.1.

The experiments above demonstrate that mechanical disruption permits PsVinfection, treatment with N-9 potentiates infection, and treatment withcarrageenan inhibits infection. The example below demonstrates onemethod that can be used to couple a label to a pseudovirus.

Example 3 Coupling of Alexa Fluor 488 Dye to Pseudovirions

Coupling of Alexa Fluor 488 dye to HPV16-RFP pseudovirions was performedaccording the manufacturer's instructions for protein labeling (A10235,Molecular Probes). The dye-coupled capsids were purified by gelfiltration over a column of 2% 50- to 150-μm agarose beads (Agarose BeadTechnologies). Re-titering of the dye-conjugated pseudovirionpreparation confirmed that its infectivity remained comparable to thatof nonlabeled pseudovirus.

The experiment above shows that labels can be successfully coupled topseudoviruses and the infectivity of labeled pseudoviruses arecomparable to that of nonlabeled pseudoviruses. The next example belowdemonstrates a technique for imaging labels coupled to or expressed inpseudoviruses.

Example 4 Multispectral Fluorescence Imaging and Statistical Analysis

To generate a more quantitative assay for cervicovaginal infection, amethod for measuring the total reporter gene expression in whole tissuesamples using a multispectral fluorescence imaging device was developed.For these analyses, the entire mouse vagina and cervix were assessed forreporter gene expression, which generated data on the distribution andintensity of infection and the mean intensity per pixel, thus allowingquantitative comparison between specimens (FIG. 2).

The reproductive tract of each mouse, from the external genitalia to thelower half of the uterine horns, was excised and stored in PBS on icefor <6 h before imaging. A Maestro (CRi, Woburn, Mass.) imaging devicewith a green excitation filter and a 580-nm long-pass emission filterwas used to obtain images from 550 nm to 9.00 nm in 10-nm wavelengthincrements. Using the spectral signature of RFP in infected tissues assignal and the background autofluorescence in uninfected tissues asnoise, a spectral unmixing algorithm was applied to the composite imagesto determine the intensity and location of infection. The open-sourcesoftware Image J, available online, was used to calculate the meansignal per pixel in a region of interest (ROI) in the grayscalerepresentation of unmixed signal. The mean of the numbers thus generatedrepresents the result of each particular experimental condition. In somecases, to determine whether the difference between these means wasstatistically significant, an unpaired Student's t-test was performedand the results reported in the text as a P value.

Conceptrol-treated mice were mock infected or challenged withHPV-16-tdTomato pseudovirus. After 3 days, the entire reproductive tractwas dissected out and the ventral wall of the vagina and cervix incisedsagitally. Composite Maestro image with unmixing algorithm was applied.Red signal represented the location of infection compared to backgroundautofluorescence. Unmixed tdTomato signal was converted to grayscale.ImageJ analysis.

Mean signal per pixel within the ROI was computed. For mock treatedmice, the ROI Area was 3.3×10⁷ and the mean signal per pixel was 21.4.For pseudovirus-challenged mice, the ROI Area was 3.5×10⁷ and the meansignal per pixel was 1316.1.

Example 5 The Intact Cervicovaginal Mucosa is Resistant to HPV InfectionMethods of N-9/Carrageenan Example

Red Fluorescent Protein (RFP) PsV was used to study papillomavirusinfection of the mouse genital tract. Surprisingly, it was found thatthe intact genital tract was completely resistant to infection afterdeposition of 10⁷ infectious units into the vagina or endocervicalcanal. (FIG. 3) Even more surprising, green fluorescent dye-coupledvirus bound neither the squamous or simple epithelium that lines thefemale reproductive tract. (FIG. 4)

Example 6 HPV Pseudoviruses do not Infect Intact Normal Tissue

HPV16 pseudovirions containing the RFP expressing plasmid (approximately10⁸ tissue culture infectious units) was administered atraumaticallyonto the following tissue surfaces: oropharygeal mucosa, tongue, smallintestines, large intestine, anal canal, eye conjunctiva, trachea,bronchi, parietal peritoneum, gastrointestinal tract serosa,gastrointestinal tract mesentery, liver, spleen, bladder, uterus,ovaries, external skin and lung parenchyma. Infection, as assessed byred fluorescence using confocal microscopy, was only observed in thelung parenchyma. The following example demonstrates that pseudovirusesselectively infect cancer cell lines.

Example 7 HPV Pseudoviruses Infect Many Human Tumor-Derived Cell Lines

A panel of 59 human tumor cell lines was obtained from the NationalCancer Institute's Developmental Therapeutics Program (DTP) In VitroCell Line Screening Project (IVCLSP), for the purpose of testinginfectability by HPV pseudoviruses (FIGS. 5 and 6). HPV5 and HPV16pseudovirions were chosen for the screen as representative of cutaneousand mucosatropic HPVs, respectively. The purified infectiouspseudoviruses containing GFP reporter plasmids were prepared asdescribed in Buck, C. B., Pastrana, D. V., Lowy, D. R, Schiller J. T.Generation of HPV pseudovirions using transfection and their use inneutralization assays. Methods Mol. Med. 119:445-462, 2005, which ishereby expressly incorporated by reference in its entirety. For HPV5,plasmids p5L1w, p5L2w and pfwb were used; and or HPV16, p16shell andpfwb plasmids were used. Purified pseudoviruses were titered on 293TTcells as described in Buck et al. above, and titer of stocks determinedto be 1.4×10E9 infectious units/ml for HPV16 and 2.2×10E7 infectiousunits/ml for HPV5.

The 59 human tumor cell lines from the DTP were inoculated into 96 wellflat-bottomed microtiter plates in 100 μL at plating densities rangingfrom 5,000 to 40,000 cells/well depending on the doubling time ofindividual cell lines.

In addition, HeLa, a human cervical epithelial cell line, (catalog#CCL-2, ATCC, Manassas, Va. 20108) was inoculated the same way as thetumor cell lines at 5,000 cells/well. All cell lines were grown in RPMI1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. Aftercell inoculation, the microtiter plates were incubated at 37° C., 5%CO2, 95% air and 100% relative humidity for 24 hours prior to additionof pseudovirus. Three, ten-fold serial dilutions were made of eachpseudovirus in DPBS+0.8M NaCl. Five μl of each dilution, undilutedpseudovirus and DPBS+0.8M NaCl (background) were added into duplicatewells for each cell line. Plates were incubated at 37° C., 5% CO2, 95%air and 100% relative humidity for 24 hours prior to addition of 150μl/well RPMI 1640 medium containing 5% fetal bovine serum and 2 mML-glutamine. Plates were incubated for another 48 hours prior to FACSanalysis (72 hour total infection time). The cells from each well wereharvested separately and subject to FACS analysis. The percent of GFPpositive cells was determined for each sample. Values obtained fromduplicate samples were averaged and the background was subtracted. Thedilution of pseudovirus that produced 1-10% GFP positive cells (in thelinear range of the FACS analysis) was used to calculate the HPV5 andHPV16 virus titers. As shown in FIGS. 5 and 6, the panel of tumor celllines were permissive for HPV5 and 16 pseudovirus infection. Theexperiments above indicate that papilloma pseudoviruses specificallyinfect cancer cell lines. The next example demonstrates thatpseudoviruses selectively infect tumor cells in vivo.

Example 8 HPV Pseudoviruses Preferentially Infect Tumor Cells in APeritoneal Tumor MetastasisModel

An established murine ovarian cancer tumor model was used to test theefficiency and specificity of pseudovirus infection of peritoneal tumornodule implants. This model uses SHIN3-DSR¹, which is a human ovariancancer cell line stably transfected with a red fluorescent protein (RFP)plasmid so that RFP is constitutively expressed in the tumor cell (FIG.7). Intraperitoneal tumor xenografts were established in female nudemice 14 days after i.p. injection of 2×10⁶ SHIN3-DSR's in 200 μl ofsterile PBS. Three days after i.p. injection of 5×10⁹ infectious units(IU) of HPV16-GFP in 300 μl of sterile PBS, the mice were euthanized andthe peritoneal membranes were analyzed by multispectral fluorescenceimaging. The anatomy of the imaged tissue and the conceptual frameworkfor the imaging are similar to that found in Reference 1, FIG. 4c .Specifically, a portion of the peritoneal membrane of the gut mesenterywas selected at random and spread out on a nonfluorescent plate. Twoseparate composite images were obtained with a Maestro imaging device.For the first, a band-pass filter from 445 to 490 nm and a long-passfilter over 515 nm were used for emission and excitation light,respectively. The tunable filter in the Maestro was automaticallystepped up in 10-nm increments from 500 to 800 nm while the cameracaptured images at each wavelength interval with a constant exposure.For the second, the band pass and longpass filter were 503 to 555 and580, respectively, and the 10 nm wavelength increments ranged from 500to 800. A spectral unmixing algorithm was applied to the first image toobtain an unmixed image of GFP and of autofluorescence. A separatealgorithm was applied to the second image to obtain an unmixed image ofRFP and of autofluorescence. These two final images are displayed, alongwith the overlay, demonstrating a high degree of colocalization ofsignal. Following multispectral imaging, these tissues were snap frozen,sectioned on a cryotome and analyzed by confocal microscopy. Thisconfirmed HPV16-GFP infection by demonstration of GFP expression inRFP-expressing SHIN3-DSR tumor nodules. In addition, confocal analysisshowed minimal, if any, pseudovirus infection of adjacent normalperitoneal membrane. This experiment indicated that HPV16 pseudovirusefficiently infected ovarian cancer cells implanted on the peritonealmembrane (FIG. 8). It further showed that infection was highly specificfor tumor cells, with normal peritoneal surfaces spared from infection.

Similar results were obtained in a murine model that used SKOV3, a humanovarian cancer cell line, were injected (FIG. 9). Just as above,Intraperitoneal tumor xenografts were established in female nude mice 14days after i.p. injection of 1×10⁵ SKOV3 cells. Three days after i.p.injection of 1×10⁸ infectious units (IU) of HPV16 PsV-luciferase orHPV16 PsV-RFP, the mice were euthanized and the peritoneal membraneswere analyzed by multispectral fluorescence imaging as discussed above.

HPV16-Luciferase

No signal was observed in control mice that lacked both HPV16PsV-luciferase and tumors, but where substrate (d-luciferin 120 mg/kgi.p.) was added. In addition, no signal was observed in control micethat lacked tumors, but received HPV16-luciferase and substrate. In micethat possessed tumors, HPV16-luciferase, and substrate, a significantsignal was observed. (FIG. 10)

HPV16-RFP

No signal was observed in control mice that were injected with HPV16PsV-RFP but lacked tumors. In mice that possessed tumors and receivedHPV16 PsV-RFP, significant fluorescence was observed. (FIGS. 11 and 12).

These experiments confirmed the finding that HPVI6 pseudovirusefficiently infected ovarian cancer cells implanted on the peritonealmembrane. It further confirmed that infection was highly specific fortumor cells, with normal peritoneal surfaces spared from infection.

Example 9 HPV Pseudovirus-Mediated Suicide Gene Therapy of OvarianCarcinoma

Based on NCI-60 human tumor-derived cell line survey, and on the resultsof the experiment described above, it is contemplated thatintraperitoneal delivery of pseudovirus will result in efficient andspecific infection of ovarian cancer cells confined to the peritonealcavity, and that this method can be used to treat ovarian cancers. Byone approach a method of suicide gene therapy, in which transduction ofthe gene for herpes simplex thymidine kinase (TK) is followed bysystemic treatment with the prodrug, gangcyclovir. In both the SHIN3nude mouse model, and in the MOSEC syngeneic, immunocompetent mousemodel for ovarian cancer, it is contemplated that xenograftedintraperitoneal tumor cells will be efficiently transduced by HPV16-TKpseudovirus, and that TK-expressing tumor cells will convertsystemically administered gangcyclovir to its toxic triphosphatemetabolite, killing tumor cells. This will result in a therapeuticresponse, as measured by decreased tumor burden and increased survivaltime in xenografted mice. Alternatively pseudovirus expressing the oligoT will be used to induce regression of the tumor cells. Furthermore,based on the NCI-60 cell line experiments, it is contemplated that freshexplants of human ovarian carcinomas will be permissive to pseudovirusinfection. This phenomenon is illustrated using a protocol similar toone previously described, in which the Krumdieck thin-slice tissueculture system is used to prepare thin sections of cancer nodulessuitable for analysis of virus transduction ex vivo. (See e.g., 1. Hama,Y. et al. A target cell-specific activatable fluorescence probe for invivo molecular imaging of cancer based on a self-quenchedavidin-rhodamine conjugate. Cancer Res 67, 2791-9 (2007) and 2. Kirby,T. O. et al. A novel ex vivo model system for evaluation ofconditionally replicative adenoviruses therapeutic efficacy andtoxicity. Clin Cancer Res 10, 8697-703 (2004)).

Example 10 Oligo T Expressing HPV Pseudoviruses for CombinedCytotoxicity and Immunity to Tumors

Oligo T expressing pseudoviruses were prepared in order to provide asimple approach to combine anti-tumor therapeutic vaccines andanti-tumor cytotoxic gene therapy.

Construction of pPolyT plasmid:

Two Oligos

(GCGGCGTCTAGAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTT; SEQ IDNO:1) and GCGGCGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A AAAAAA;SEQ ID NO: 2) were snap-annealed, then extended with T4 DNA polymerase(New England Biolabs). The resulting duplexed DNA was digested with XbaI and ligated into the Xba I site of a murine pol-I promoter/terminatorconstruct, p417-Ron (obtained from Ron A M. Fouchier, Erasmus MedicalCenter, the Netherlands). Clones were sequenced, and a clone with a 45basepair T tract on the sense strand was selected.

Experiment Documentation of pPolyT Cytotoxicity:

HaCaT cells were transfected with pBluescript II KS+ (Stratagene) orpPolyT using Lipofectamine LTX (Invitrogen). 48 hours later, the cellswere inspected by light microscope and treated with a colorimetricmetabolic substrate (WST-1, Roche). Cells receiving pPolyT exhibitedreticulated morphology and were quite sparse relative to pBluescripttransfected (or untransfected) cells. Turnover of WST-1 was reducedby >2-fold in the pPolyT transfected cells.

Example 11 Intravenous Administration of HPV Pseudovirus Targets LungMetastases

According to an established protocol for producing lung tumor metastases(see, e.g., Qian et al. Prophylactic, therapeutic and anti-metastaticeffects of an HPV-16 mE6Δ/mE7/TBhsp70Δ fusion protein vaccine in ananimal model. Immunology Letters 102(2):191-201 (2006)), a series ofmice were injected with 2×10⁴ TC-1 cells intravenously 2 weeks prior tothe experiment. All mice were then treated with approximately 1×10⁷HPV16 PsV-luciferase intravenously, including the control animals, whichreceived saline instead of tumor cells. Luciferase activity wassubsequently measured upon introduction of substrate. No signal wasdetected in the control mice. In the mice that had been inoculated withtumor cells, significant signal was detected, indicating thepseudoviruses efficiently targeted lung metastases (FIG. 13). Thisexperiment demonstrated that intravenous administration of pseudovirusresults in specific targeting of tumor cells and sparing of normaltissue, similar to the pattern demonstrated after administration byother routes.

Example 12 Diagnosis and Treatment of Cervical Cancer

A subject having cervical cancer is provided a composition comprising apapilloma VLP coupled to a radionuclide (e.g., ³H, ¹²⁵I, ¹³¹I, ³²P, ³⁵S,¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹¹At, ²¹²Pb, ⁴⁷Sc,¹⁰⁹Pd, ¹⁸⁶Re, ¹⁸⁸Re, or ²¹²Bi). The composition can be provided in anamount of approximately 0.1 mg to approximately 10 mg. Alternatively,the composition can be provided in an amount sufficient to deliver 100,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500,7000, 7500, 8000, 8500, 9000, 9500, or 10000 rads to the cancer cells.The composition can be provided in a convenient vaginal application andthe composition may be in a liquid or gel form. The compositioncomprising the papilloma VLP coupled to the radionuclide is provided tothe subject and the labeled VLPs are allowed to bind to the cervicalcancer cells for 2, 4, 6, or 8 hours. After binding, the unbound labeledVLPs are removed by successive lavage. The amount of radioactivitypresent in the vaginal canal is assessed by dosimetry determinations(e.g., dosimetry badges). A new badge is provided daily for 4 weeks andthe badges are collected at the end of the evaluation and a gradualdecrease in radioactive exposure will be seen over time.

It is also envisioned that a weekly cervical biopsy will be taken so asto evaluate the histological reduction of cancer cells during theexperiment. The results will show that the radiolabeled VLPs selectivelybound and identified the cervical cancer cells in the subject's vaginalcanal and, overtime, the radioactivity contributed to the reduction ofthe presence and proliferation of the cancer cells and the restorationof normal cell morphology, as compared to untreated control subjects.

What is claimed is:
 1. A method comprising administering to cancer cellsin a subject, a papilloma pseudovirus that comprises a fluorescent dyeor a papilloma virus-like particle (VLP) that comprises a fluorescentdye, and exposing the fluorescent dye in the cancer cells in the subjectto an excitation wavelength of light.
 2. The method of claim 1, whereinthe fluorescent dye is chemically coupled to the pseudovirus or to theVLP.
 3. The method of claim 2, wherein the fluorescent dye is chemicallycoupled to the surface of the papilloma pseudovirus or to the surface ofthe papilloma VLP.
 4. The method of claim 1, wherein the papillomapseudovirus or the papilloma VLP is administered to cancer cells in aneye in the subject.
 5. A method comprising administering to cancer cellsin an eye of a subject, a papilloma pseudovirus chemically coupled to afluorescent dye or a papilloma virus-like particle (VLP) chemicallycoupled to a fluorescent dye, and exposing the fluorescent dye in thecancer cells in the subject to an excitation wavelength of light.
 6. Themethod of claim 5, wherein the fluorescent dye is chemically coupled tothe surface of the papilloma pseudovirus or to the surface of thepapilloma VLP.
 7. The method of claim 1, wherein the papillomapseudovirus or the papilloma VLP is injected into the cancer cells inthe subject.
 8. The method of claim 1, wherein the papilloma pseudovirusor the papilloma VLP is injected into cancer cells in an eye in thesubject.